Recently, a large number of proteins, polypeptides, synthetic compounds, and compounds extracted from natural resources having physiological activity and the application thereof to pharmaceuticals has been extensively studied. However, these physiologically active substances have short half-lives in blood when they are injected to a body and hence it is difficult to obtain a sufficient pharmacological effect. This is because the physiologically active substances injected to a body are usually cleared from the body because of the filtration through glomeruli in the kidney and the uptake by macrophages in the liver and spleen. Therefore, it is attempted to improve the behavior in a body by including these physiologically active substances in liposomes or polymer micelles or increasing their molecular weight through chemical modification with polyethylene glycol which is an amphiphatic polymer. Polyethylene glycol exhibits a low interaction with the other bio-components owing to its steric repulsion effect and as a result, proteins and polypeptides such as enzymes modified with polyethylene glycol exhibit an effect of avoiding the filtration through glomeruli in the kidney and bio-reactions such as immunoreaction, so that they achieve half-lives in blood longer than those of unmodified substances. Moreover, they also have decreased toxicity and antigenicity and further exhibit an effect of enhancing the solubility of a sparingly water-soluble compound having a high hydrophobicity.
Hitherto, in the case of modifying a physiologically active substance with polyethylene glycol, particularly in the case of modifying a low-molecular-weight drug or peptide, there arises a problem that few reactive functional groups can be used for the modification with polyethylene glycol. Furthermore, when a peptide or drug is modified with many polyethylene glycol molecules for obtaining a sufficient effect of the modification with polyethylene glycol, the active site of the peptide or drug is blocked and hence problems may arise that its own function and efficacy cannot be exhibited sufficiently and enough solubility in water cannot be obtained.
For solving such problems, the reduction of the number of modification with polyethylene glycol using a branched polyethylene glycol derivative has been attempted. JP-B-61-42558 proposes a polyethylene glycol-modified L-asparaginase. However, cyanuric chloride as a starting material for a reactive polyethylene glycol derivative has three reactive sites and hence it is difficult to introduce two polyethylene glycol chains thereinto selectively. Accordingly, it is difficult to synthesize a highly pure polyethylene glycol-modified L-asparaginase.
Also, JP-A-10-67800 proposes a polyethylene glycol-modified interferon α. However, this substance has three urethane and amide bonds including the linkage between interferon α and the poly(ethylene glycol) oxy group. These bonds are labile to hydrolysis during storage or during the reaction under an alkaline condition and as a result, there arises a problem that the branched polyethylene glycol moiety is decomposed to a single chain. This is because the polyethylene glycol derivative which is the intermediate material has been produced by a method wherein two monomethoxypolyethylene glycols and amino groups at α- and ε-positions of lysine are combined through urethane bonds and then the carboxyl residue of lysine is converted into a succinimide ester. In addition, in order to produce the polyethylene glycol-modified interferon α, there arises a problem that increased impurities are produced owing to the multi-step process, such as the activation of the terminal hydroxyl groups of two monomethoxypolyethylene glycols, the combination with lysine, the activation of the carboxyl residue of lysine, and the combination with interferon α.
Accordingly, it is desired to develop a bio-related substance formed by highly stable bonds, a process for producing the same, and a branched reactive polyalkylene glycol derivative which can be produced in a convenient manner and in a high purity and has a higher stability.